Bremer A, Henn C, Goldie K N, Engel A, Smith P R, Aebi U
M.E. Müller-Institute for Microscopy, Biozentrum University of Basel, Switzerland.
J Mol Biol. 1994 Oct 7;242(5):683-700. doi: 10.1006/jmbi.1994.1617.
We have recorded dark field images of negatively stained F-actin filaments polymerized with 2 mM MgCl2 and 50 mM KCl with a scanning transmission electron microscope and computed 3-D reconstructions using a helical parameter search to optimize simultaneously the helical repeat length, the radial position of the filament axis, and the helical selection rule. The resulting optimized averaged filament 3-D reconstruction at 2.5 nm resolution is remarkably similar to an atomic model of the F-actin filament. By comparison, several structural features of the reconstruction can be interpreted at the level of distinct secondary structure elements, and predictions made by the atomic model could be verified: for instance, the density connecting the two long-pitch helical strands in our reconstruction co-localizes with an extended beta-hairpin, the "hydrophobic loop" (i.e. residues 262 to 274), which according to the atomic model establishes the major intersubunit contact between the two long-pitch helical strands. The most pronounced structural variations among individual filament 3-D reconstructions were observed in (1) the details of the intersubunit contact pattern between the two long-pitch helical strands, and (2) the exact size and shape of subdomain 2 of the F-actin molecule, which appears rather flexible and easily deformed. In addition, we found that all phenotypes of F-actin filament 3-D reconstructions that arise from small deviations from the optimal helical parameters or from lowering the nominal resolution exhibited stronger intersubunit contacts between than along the two long-pitch helical strands, a structural feature that has been emphasized for a number of F-actin filament 3-D reconstructions in the past. Since this is clearly at variance with the relative strength of the intersubunit contacts as predicted by the atomic model, it may represent an artifactual structural feature arising from low-resolution data or suboptimal helical data processing, and should therefore be interpreted with caution in terms of indicating chemical, mechanical or conformational states of the F-actin filament.
我们使用扫描透射电子显微镜记录了在含有2 mM氯化镁和50 mM氯化钾的条件下聚合的负染F-肌动蛋白丝的暗场图像，并使用螺旋参数搜索进行三维重建，以同时优化螺旋重复长度、丝轴的径向位置和螺旋选择规则。所得的在2.5纳米分辨率下优化的平均丝三维重建与F-肌动蛋白丝的原子模型非常相似。相比之下，重建的几个结构特征可以在不同二级结构元件的层面上进行解释，并且原子模型所做的预测可以得到验证：例如，我们重建中连接两条长间距螺旋链的密度与一个延伸的β-发夹结构“疏水环”（即残基262至274）共定位，根据原子模型，该结构建立了两条长间距螺旋链之间的主要亚基间接触。在单个丝三维重建中观察到的最明显的结构变化出现在：（1）两条长间距螺旋链之间亚基间接触模式的细节，以及（2）F-肌动蛋白分子亚结构域2的确切大小和形状，该亚结构域显得相当灵活且容易变形。此外，我们发现，由于与最佳螺旋参数的小偏差或降低标称分辨率而产生的F-肌动蛋白丝三维重建的所有表型，在两条长间距螺旋链之间表现出比沿链更强的亚基间接触，这是过去许多F-肌动蛋白丝三维重建中所强调的一个结构特征。由于这显然与原子模型预测的亚基间接触的相对强度不一致，它可能代表了由低分辨率数据或次优螺旋数据处理产生的人为结构特征，因此在用于指示F-肌动蛋白丝的化学、机械或构象状态时应谨慎解释。
